There exists a never ending demand for additional and more accurate information about biological objects, such as cells, in humans, animals, plants and other organisms. Cells are very thin and contain only very few structures that are easily identifiable using a light microscope. Several different methods have been developed to make cells and cell structures more visible. Many of the methods include stains that colour the entire cell or stains that colour only certain cell structures. These stains can be fluorescent or non-fluorescent. An example is trypan blue which is a widely used non-fluorescent stain that colours the entire cell blue. Trypan blue is only used for dead cells. The exact methods used to stain the cells vary as different stains have different properties. A rather recent labelling method is to induce the cell itself to produce a fluorescent substance such as green fluorescent protein (GFP). This labelling can be either very selective, showing a specific cell structure, or non-selective, showing the entire cell. Another widely used method called immuno-labelling is to label the cells using antibodies. Antibodies are proteins that are found in the body fluids of vertebrates. Antibodies bind to substances and particles, e.g. bacteria or viruses, which are thus recognized as foreign objects, and they target very specific structures. This is useful for biological and medical research as antibodies can be used to identify very specific small structures in a cell or parts of a cell. Antibodies are very difficult to detect, but they can be conjugated to a wide array of different molecules or structures that make them clearly visible. Different antibody conjugates are suited for different detection methods such as light microscopy, electron microscopy, spectrometry or radioactivity measurements. Conjugated antibodies can be used to bind directly to a target. If a stronger signal is needed, the signal can be amplified by first allowing unlabelled antibodies to bind to the target, and then allow a second set of conjugated antibodies to bind to the first set of non-conjugated antibodies. The first set of antibodies is called primary anti-bodies and the second set is called secondary antibodies. Further layers of antibodies can be used if the signal needs more amplification.
Different types of stains, labels and antibody conjugates have different light-related characteristics such as the ability to absorb light at several or a few wavelengths, to emit light at certain wavelengths, to disperse light or to polarize light. These different characteristics allow the stains to show as colouring or fluorescence when using a light microscope, a phase contrast microscope, a fluorescence microscope or a flow cytometer. The stains, labels or conjugates can also be measured using e.g. a spectrophotometer or a fluorescence plate reader.
Examples of antibody conjugates which render absorption differences are quantum dots, plastic balls, glass beads and semiconductors. Examples of antibody conjugates which scatter light or polarize it are dynabeads, gold particles, nano crystals or magnetic beads.
Digital holographic microscopy enables the study of living cells without the need for markers. In WO2009/154558, which describes an observation vessel for digital holographic microscopy and a method for digital holographic microscopy by use of said vessel, there is disclosed that digital holographic microscopy enables studies of living cells without the need for markers or stains. Fact is that digital holographic microscopy and imaging renders monochromatic images, which up til now have implied that such technology has not been interesting for analysing labelled or stained cell samples. The use of markers is increasing, so also the interest in such markers and the possible applications in relation thereto for different sample analysis.
One way to study cells by means of digital holographic microscopy is disclosed in WO 2007/073345, where a method for analyzing a stage of development of cells and a device for enablement of the analysis is disclosed.
As mentioned above, another method for studying cells by means of digital holographic microscopy is disclosed in WO2009/154558.
As said above, the technology of digital holographic microscopy and imaging renders monochromatic images. This implies that this technology, e.g. defined in the inventions disclosed in WO 2007/073345 and WO2009/154558, has not been of interest in relation to use of labelling and staining of cell samples.